Chemical moieties that quench fluorescent light operate through a variety of mechanisms, including fluorescence resonance energy transfer (FRET) processes and ground state quenching. FRET is one of the most common mechanisms of fluorescent quenching and can occur when the emission spectrum of the fluorescent donor overlaps the absorbance spectrum of the quencher and when the donor and quencher are within a sufficient distance known as the Förster distance. The energy absorbed by a quencher can subsequently be released through a variety of mechanisms depending upon the chemical nature of the quencher. Captured energy can be released through fluorescence or through nonfluorescent mechanisms, including charge transfer and collisional mechanisms, or a combination of such mechanisms. When a quencher releases captured energy through nonfluorescent mechanisms FRET is simply observed as a reduction in the fluorescent emission of the fluorescent donor.
Although FRET is the most common mechanism for quenching, any combination of molecular orientation and spectral coincidence that results in quenching is a useful mechanism for quenching by the compounds of the present invention. For example, ground-state quenching can occur in the absence of spectral overlap if the fluorophore and quencher are sufficiently close together to form a ground state complex.
Quenching processes that rely on the interaction of two dyes as their spatial relationship changes can be used conveniently to detect and/or identify nucleotide sequences and other biological phenomena. For example, the change in fluorescence of the fluorescent donor or quencher can be monitored as two oligonucleotides (one containing a donor and one containing a quencher) bind to each other through hybridization. Advantageously, the binding can be detected without separating the unhybridized from the hybridized oligonucleotides.
Alternatively, a donor and quencher can be linked to a single oligonucleotide such that there is a detectable difference in fluorescence when the oligonucleotide is unhybridized as compared to when it is hybridized to its complementary sequence. For example, a self-complementary oligonucleotide designed to form a hairpin can be labeled with a fluorescent donor at one end and a quencher at the other end. Intramolecular annealing can bring the donor and quencher into sufficient proximity for FRET and fluorescence quenching occurs. Intermolecular annealing of such an oligonucleotide to a target sequence disrupts the hairpin, thereby increasing the distance between the donor and quencher, and resulting in an increase in the fluorescent signal of the donor.
Oligonucleotides labeled in a similar manner can also be used to monitor the kinetics of PCR amplification. In one version of this method the oligonucleotides are designed to hybridize to the 3′ side (“downstream”) of an amplification primer so that the 5′-3′ exonuclease activity of a polymerase digests the 5′ end of the probe and cleaves off a dye (either the donor fluorophore or quencher) from that end. The fluorescence intensity of the sample increases and can be monitored as the probe is digested during the course of amplification.
Similar oligonucleotide compositions find use in other molecular/cellular biology and diagnostic assays, such as in end-point PCR, in situ hybridizations, in vivo DNA and RNA species detection, single nucleotide polymorphism (SNPs) analysis, enzyme assays, and in vivo and in vitro whole cell assays.
As noted previously, the energy transfer process requires overlap between the emission spectrum of the fluorescent donor and the absorbance spectrum of the quencher. This complicates the design of probes because not all potential quencher/donor pairs can be used. For example, the quencher BHQ-1, which maximally absorbs light in the wavelength range of about 500-550 nm, can quench the fluorescent light emitted from the fluorophore fluorescein, which has a wavelength of about 520 nm. In contrast, the quencher BHQ-3, which maximally absorbs light in the wavelength range of about 650-700 nm would be less effective at quenching the fluorescence of fluorescein but would be quite effective at quenching the fluorescence of the fluorophore Cy5 which fluoresces at about 670 nm. The use of varied quenchers complicates assay development because the purification of a given probe can vary greatly depending on the nature of the quencher attached.
Many quenchers emit energy through fluorescence reducing the signal to noise ratio of the probes that contain them and the sensitivity of assays that utilize them. Such quenchers interfere with the use of fluorophores that fluoresce at similar wavelength ranges. This limits the number of fluorophores that can be used with such quenchers thereby limiting their usefulness for multiplexed assays which rely on the use of distinct fluorophores in distinct probes that all contain a single quencher.
Thus, new compositions are needed that quench fluorescence over a broad spectrum of wavelengths such that a single quencher can be used with a broad range of fluorophores. Ideally, such quenchers will not fluoresce so that the background fluorescence of probes is minimized giving such probes the potential to be more sensitive and more useful in multiplexed assays. The ideal quenchers should also have physical properties that facilitate their purification and the purification of probes into which they are incorporated. Such quenchers should also be chemically stable so that they can be incorporated into biological probes and used in assays without significant degradation. Ideally, such probes will be suitable for direct use in the synthesis of DNA oligomers so that oligonucleotides can be synthesized to contain them, as opposed to chemically adding the quencher to an oligonucleotide postsynthetically. Nevertheless, the quenchers should contain suitable reactive moieties to provide for their convenient incorporation into biologically relevant compounds such as lipids, nucleic acids, polypeptides, and more specifically antigens, steroids, vitamins, drugs, haptens, metabolites, toxins, environmental pollutants, amino acids, peptides, proteins, nucleotides, oligonucleotides, polynucleotides, carbohydrates, and the like. Lastly, the most useful compositions should be easily manufactured.
The invention provides nonfluorescing compositions with strong fluorescence quenching properties that function over a surprisingly wide wavelength range. Consequently, the disclosed compositions exhibit lower fluorescent backgrounds than quenchers that quench light at certain wavelengths and emit fluorescence at nearby wavelengths. Moreover, the anthraquinone quenchers of the present invention can be easily manufactured and purified. The compositions can be incorporated into biologically relevant compounds and, in many cases, impart useful purification properties to these compounds. These and other advantages of the invention, as well as additional inventive features, will be apparent from the description of the invention provided herein.